P419: IDENTIFICATION OF NOVEL THERAPEUTIC OPTIONS FOR VENETOCLAX-RESISTANT AML CELLS THROUGH DRUG REPURPOSING
Authors: A. Ladungova, D. Busa, Y. Lodhi, J. Hyl, M. Culen, M. Smida
Journal: HemaSphere (2022)

Background: Acute myeloid leukemia (AML) is a malignant disease derived from the bone marrow precursors of myeloid lineage. Treatment options are rather limited, primarily based on chemotherapy, and often result in disease progression. Recently, venetoclax-based therapies have transformed the frontline regimens of elderly patients and patients unfit for intensive chemotherapy. Despite its promising outcomes in clinical studies, multiple resistant subclones evolved during the treatment acting as a barrier in disease regression. Understanding the venetoclax-resistance mechanisms and detecting the major determinants could reveal previously unrecognized novel perspectives for therapeutic strategies to improve patients’ outcomes. Moreover, performing high-throughput screenings with clinically approved drugs could reveal novel treatment options for resistant subclones of AML. Aims: Our project aims 1) to identify novel FDA-approved drugs effective for treating venetoclax-resistant AML cells through a high throughput screening, 2) to validate the most effective compounds, prove their specificity, and 3) to investigate the molecular background mechanisms underlying the observed sensitivity. Methods: Two different models mimicking the microenvironment of venetoclax-resistance formation have been established: 1.) Patient-derived mouse xenograft (PDX) model. AML PDX model was generated by using AML patient sample transplanted into an immunocompromised murine host and treated with different regimens of venetoclax; 2.) Venetoclax-resistant cell lines (MOLM-13, OCI-AML3, HL-60). Generation of the venetoclax-resistant AML cell lines was achieved through the chronic administration of the compound, inhibiting the cell viability to 80-90% in multiple rounds with gradually increasing concentrations until the cells become fully resistant. Both models are next used for the high-throughput screening of 859 approved drugs, covering broad chemical space. The standard concentration (1 μM) of drugs in the library is automatically added to cells on 384-well-plates with a programmed liquid handling system epMotion (Eppendorf). Cell viability is determined after 72 hours by a Cell-titer Glo assay. Compounds with the strongest effect upon resistant AML cell viability are validated through 10-point dose-response curves with 2-fold-dilutions. Moreover, the investigation of molecular mechanisms responsible for the sensitization of resistant AML cells is planned by using various cell biology and molecular biology techniques. Results: Treating the PDX mouse model with venetoclax for 6 weeks resulted in the generation of resistant cells. Importantly, these cells also acquired additional resistance to topoisomerase inhibitors, DNA-damaging agents and many other compounds. On the contrary, both 6-week as well as 3-week venetoclax treatments generated cells exquisitely sensitive to the iron chelator deferoxamine mesylate and a purine nucleoside analogue clofarabine. In addition, we generated a venetoclax-resistant MOLM-13 cell line with 1500-fold decreased sensitivity and have subjected it to the drug screening to correlate its response with the PDX model. Summary/Conclusion: The outcomes of this research project point out the importance of novel treatment options to overcome venetoclax-resistance in AML cells. Further validations and investigation of the molecular mechanisms are planned for the top-performing drugs to strengthen the translational potential for AML therapy. This project was supported by the grants number MUNI/A/1330/2021, MUNI/A/1419/2021 and by OPRDE (No.CZ.02.2.69/0.0/0.0/19_073/0016943).